Detection of bioagents using a shear horizontal surface acoustic wave biosensor

ABSTRACT

Viruses and other bioagents are of high medical and biodefense concern and their detection at concentrations well below the threshold necessary to cause health hazards continues to be a challenge with respect to sensitivity, specificity, and selectivity. Ideally, assays for accurate and real time detection of viral agents and other bioagents would not necessitate any pre-processing of the analyte, which would make them applicable for example to bodily fluids (blood, sputum) and man-made as well as naturally occurring bodies of water (pools, rivers). We describe herein a robust biosensor that combines the sensitivity of surface acoustic waves (SAW) generated at a frequency of 325 MHz with the specificity provided by antibodies and other ligands for the detection of viral agents. In preferred embodiments, a lithium tantalate based SAW transducer with silicon dioxide waveguide sensor platform featuring three test and one reference delay lines was used to adsorb antibodies directed against Coxsackie virus B4 or the negative-stranded category A bioagent Sin Nombre virus (SNV), a member of the genus Hantavirus, family Bunyaviridae, negative-stranded RNA viruses. Rapid detection (within seconds) of increasing concentrations of viral particles was linear over a range of order of magnitude for both viruses, although the sensor was approximately 50×10 4 -fold more sensitive for the detection of SNV. For both pathogens, the sensor&#39;s selectivity for its target was not compromised by the presence of confounding Herpes Simplex virus type 1. The biosensor was able to detect SNV at doses lower than the load of virus typically found in a human patient suffering from hantavirus cardiopulmonary syndrome (HCPS). Further, in a proof-of-principle real world application, the SAW biosensor was capable of selectively detecting SNV agents in complex solutions, such as naturally occurring bodies of water (river, sewage effluent) without analyte pre-processing.

RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.provisional application Ser. No. 60/900,416, filed Feb. 9, 2007, Ser.No. 60/926,827, filed Apr. 30, 2007 and Ser. No. 61/009,656, filed Dec.31, 2007, each of which applications is incorporated by reference in itsentirety herein.

FIELD OF THE INVENTION

The present invention relates to the use of a shear horizontal surfaceacoustic wave biosensor adapted to detect a large number of biologicalor chemical agents, including viruses, virus fractions (membranes,biomarkers, etc.) and other microbes (prions, eukaryotic and prokaryoticcells, including fungus and bacteria) amino acid based biological agentsincluding polypeptides, DNA and RNA, lipids (including glycosylated,phosphorylated, acetylated lipids) and synthetic chemicals, includingnerve gas and other chemical agents. In the present invention, apiezoelectric material, in particular, a lithium tantalate (LiTaO₃)wafer is modified to provide on its surface at least one ligand andpreferably, a plurality of ligands, which bind to one or more biologicalagents to be detected by the biosensor. In the present invention, abiosensor to which is bound a free (unbound) ligand, emits acharacteristic wave which can be readily measured—and if the ligandbecomes bound to a biological agent, the ligand-biological agent willproduce a modified wave pattern which can be measured and result in thequalitative identification (including, in certain aspects, quantitativeor concentration information) of the biological agent in a sample. Themethod and apparatus are adaptable to identify a large number ofbiological agents as otherwise described in detail herein.

Grant Support

This work was conducted with support from the National ScienceFoundation, grant number IIS-0434120, and from the National Institute ofAllergy and Infectious Diseases, grant numbers UO1 AI56618 and UO1AI054779.

BACKGROUND OF THE INVENTION

Recently, there has been a heightened interest in developing rapid andreliable methods of detection of micro-organisms involved inbioterrorism, food poisoning, and clinical problems. Biosensors aredevices under intense development to achieve these goals and a number ofdifferent types of transduction modes are currently investigated,including electrochemical, optical, thermal, and acoustic (Deisingh,2004). Shear horizontal surface acoustic wave (SH-SAW) devices that arebased on horizontally polarized surface shear waves (HPSSW) enablelabel-free, sensitive and cost-effective detection of biomolecules inreal time and have been used for the detection of bacteria and viral DNA(Berkenpas et al., 2006; Branch and Brozik, 2004; Moll et al., 2007). ASAW device typically has a planar electrode structure consisting of apiezoelectric substrate containing inter-digital transducers (IDTs)(Branch and Brozik, 2004). An often used substrate compound that meetsmany of the required conditions for successful HPSSW generation islithium tantalate (LiTaO₃) (Branch and Brozik, 2004; Martin et al.,2004). Applying an alternating voltage via the IDTs at high frequency(typically from 80 to 400 MHz), HPSSW are generated on the substrate.These HPSSW result in a specific resonance frequency that ischaracteristic for the substrate surface wave velocity. The frequency issensitive to measurable changes on the sensor surface, for examplecaused by specific biological interactions.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to a biological ligand based biosensorcomprising a biological ligand which is complexed (coated or tethered)directly or indirectly to the surface of a piezoelectric material of abiosensor apparatus which is combined to accommodate surface acousticwave (SAW) technology.

The biosensor of the present invention has, complexed on its surface,any number of biological ligands, which are capable of binding tobioagents which are to be identified. The biological ligands may bechosen for their ability to bind a specific bioagent, or alternatively,to bind to a group or class of bioagents, depending upon common featureswhich exist between the biological ligand which is tethered to thesurface of the biosensor apparatus and the bioagent to be identified.The biological ligand may be amino acid based, such as a polypeptide(including modified polypeptides such as glycosylated polypeptides,multimeric polypeptides, including polypeptides which are multimerizedby crosslinking devices, nanoparticles, etc., and antibodies), nucleicacid based (single stranded DNA, single stranded RNA, oligonucleotides,including modified oligonucleotides) or lipid based (includingglycosylated lipid). The biological ligand is chosen to be specific fora given bioagent such that when a bioagent comes in contact with theligand, the SAW associated with the biosensor reflects a changeindicative of the binding of the bioagent to the ligand.

The present invention is based upon a biosensor apparatus featuringsurface chemistries which are capable of transducing shear horizontalsurface acoustic waves (SH-SAW) generated by piezoelectric materials, inparticular, lithium tantalate (LiTaO₃), when connected to an electricalcircuit at defined frequencies. The technology is manufactured ontowafers that can be sectioned into functional units (“chips”). Theseunits can be applied to a SAW detection board featuring a fluidichousing and a connection of the chip via aluminum delay lines to anoutput interface with a computing device (e.g., a laptop computer orother computing device) for measurements of SAW. The biosensor apparatusis depicted in FIGS. 1 a-d.

In a method of the present invention, a sample is presented to theligand based biosensor as described above and a determination of theexistence (binding) of a bioagent in the sample which is made bydetermining a change in a surface acoustic wave emanating from thebiosensor which evidences the binding of the bioagent to the ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram depicting an antibody based virus surface acousticwave (SAW) biosensor in accordance with the present invention. Thelithium tantalate (LiTaO₃) sensor surface was coated with NeutrAvidinBiotin Binding Protein (N-Avidin) and coupled to biotinylated [B]monoclonal anti-JVB antibody for Coxsackie virus. Anti-SNV-G1glycoprotein scFv antibody for SNV detection was coupled directly.Molecular interaction between virus and antibody elicits an acousticwave leading to a change in the input frequency of 325 MHz.

FIG. 1B is a photographic plan view of a pair of substrates or sensorwafers with IDT electrodes disposed thereon for making SAW measurements,showing the sensor wafers in scale compared to a dime coin (10 UScents); four aluminum delay lines are visible; one serves as thereference and three as the test delay lines.

FIG. 1C is a photographic perspective view showing a SAW detection board(thin arrow) with the fluidic housing (thick arrow) and the outputinterface device (arrow head) to a laptop computer is shown.

FIG. 1D is a block diagram of a SAW measurement assembly for making SAWmeasurements in accordance with the present invention.

FIG. 2 is a graph showing a detected phase differential as a function oftime during operation of a SAW sensor assembly in accordance with thepresent invention. More particularly, FIG. 2 is a representative SAWbiosensor plot showing the response to 1.8×10⁴ SNV particles per μl.Output was captured using a custom LabVIEW (National Instruments;website ni.com) program. The data is reported as phase differential massshift Δφ (indicated by double-headed arrow) on the y-axis as a functionof time on the x-axis, corresponding to the maximal difference betweenbuffer-calibrated phase differential before addition of agent (arrow Aat ˜280 seconds) and after stabilization of signal (arrow B at ˜420seconds). Dotted lines indicate detection at ˜15 seconds and maximalsignal difference at ˜2 minutes after addition of the agent. Singlevertically oriented data points are visible as a function of time. Seetext for details.

FIG. 3 is a graph of phase differential shift as a function of virusconcentration, depicting the detection of Coxsackie virus B4 and SNVusing the SAW biosensor, in accordance with the present invention. Datawas acquired as described in FIG. 2 and in the text. The plots show theshifts in phase differential (degrees) on the y-axis as a function ofincreasing viral particle concentrations (in virus/μl) on the x-axis.Each data point represents an average of 2-5 measurements±standarderrors. =Coxsackie virus B4; ♦=Coxsackie virus B4+HSV-1; ▪=HSV-1 alonefor the Coxsackie virus experiments; ▴=SNV; ◯=SNV+HSV-1; □=HSV-1 alonefor the SNV experiments. The solid and dotted lines represent the bestfit models and coefficient correlations (R²) for Coxsackie virus B4 ()and SNV (▴), respectively.

FIG. 4 is a bar graph phase differential shift as a function of testsolutions, showing detection of SNV in complex solutions using the SAWbiosensor. SNV viral particles were spiked into distilled (DI) water,phosphate buffered saline (PBS), sewage effluent, or Rio Grande waterfor a final concentration of 1.8×10⁴ viral particles/μl. Data wasacquired as described in FIG. 2 and in the text. The plots show theshifts in phase differential (degrees) on the y-axis as a function ofsolution input on the x-axis. Each data point represents an average of 2measurements with ±standard errors. Stars denote statisticalsignificance (p<0.05) over background (no virus) solutions usingstudent's t-test.

DETAILED DESCRIPTION OF THE INVENTION

The following terms shall be used to describe the present invention. Ininstances where a term is not defined, then the term shall be given itsordinary meaning as understood by those of ordinary skill within thecontext of its use.

The term “effective” is used to describe, within context, an amount of acomponent or compound used in the present invention in a mannerconsistent with its intended use.

The term “ligand” or “capture molecule” shall mean a biological compoundcomprising amino acids, DNA or RNA (polynucleotides oroligonucleotides), lipids or carbohydrates which may be complexed(coated or tethered) onto the surface of a piezoelectric material in abiosensor apparatus according to the present invention. Amino acid basedligands include antibodies (monoclonal or polyclonal) and fragmentsthereof (e.g. single chain Fab antibody fragments), polypeptides (atleast 25 mer), oligopeptides (about 3 mer to about 25 mer or more),peptide multimers (palindromes, crosslinked multilmeric peptides,nanoparticles, among others). DNA or RNA ligands are polynucleotides,oligonucleotides which may be single stranded DNA, single stranded RNA,oligonucleotides (phosphate backbone, etc.,) and lipids, includingglycosylated lipids and carbohydrates, including complex carbohydrates.Although the most common type of capture molecule is an antibody, otherpeptides such as receptors, enzymes, other proteins, includingdendrimers (especially in the case of prions), nucleotides(polynucleotides and oligonucleotides, aptamers, primarily DNAmolecules, but also stable RNA (preferably, single stranded) moleculesmay be used. In addition, lipids and carbohydrates (chitosan, lectins,among others) may be used as capture molecules, depending upon thenature of the bioagent to be detected.

The ligand may be designed and used to detect primary amino acidsequences, especially including biomarkers on cell or particle surfaces(including mutations, translocations, truncations and isoforms),posttranslational modifications, phosphorylations, glycoslation,non-peptide organics (nerve gasses, alkylating agents, other organictoxins), lipids, including glycosylated, phosphorylated or acetylatedlipids, double stranded DNA or RNA (especially after denaturing) andsingle stranded DNA or RNA. The ligand may include a nucleocapsinprotein capture DNA, as well as other capture DNA and RNA molecules(such as aptamers), among others.

Ligands may be broadly used and applied. Essentially any biologicalmolecule capable of binding a bioagent as otherwise described hereinfinds use in the present invention. Exemplary ligands may be foundthroughout the literature and are numerous. An excellent directory whichprovides a large number of amino acid based ligands, especiallymonoclonal and polyclonal antibodies for use in the present invention isLinscott's directory of immunological and biological reagents, availableat linscottsdirectory.com. DNA and RNA ligands (e.g., capture DNA/RNA)which bind to bioagents may be found throughout the literature. Inapplications of the present invention, depending upon the type andactual size of the ligand to be tethered to the biosensor, thenumber/concentration of ligands which are tethered to a biosensor rangesfrom 1 to about 100,000 or more per cm², from 10 to about 50,000, about50 to about 25,000, about 100 to about 20,000, about 500 to about 15,000about 750 to about 12,500, about 1000 to about 10,000, about 1250 toabout 7500, about

Exemplary ligands include monoclonal and polyclonal antibodies,including anti-Coxsackie virus monoclonal antibody (mouse IgG2a),anti-Sin Nombre virus (SNV) G1 glycoprotein (single chain Fv, scFv fromphage display), anti-Herpes Simplex virus I (HSV-1) antibody(polyclonal), anti-αvβ3 integrin antibody, anti-Interleukin-12 (IL-12)monoclonal antibody (mouse IgG1), anti-Interleukin-6 (IL-6) polyclonal,among others; peptides, including 9 amino acid cyclic peptides from SinNombre Virus phage display as described in greater detail herein, 9amino acid cyclic peptides from Andes virus phage display as describedin greater detail herein, linear peptides including a 16 amino acidpeptide from Dengue virus phage display; nucleic acids, including SinNombre virus-N(SNV-N) and SNV-M capture DNA (analyte is Sin Nomber virus(SNV) RNA) and BRCA1 capture DNA (single-stranded, 30 mer (base units inlength)-analyte is BRCA1 DNA (single stranded-60 base units in length),among others.

The term “bioagents” or “analytes” used synonymously within context,shall mean viruses and their extracts, prions and their extracts,eurcaryotic cells, fragments and extracts, prokaryotic cells (especiallybacteria), fragments and extracts, prokaryotic spores, fragments andextracts, protein and peptide markers (biomarkers, especially biomarkerson cell surfaces), serum proteins, isolated proteins, syntheticchemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts,including mitochondria and other organelles, prokaryotic membranes, DNAviruses (single and double stranded DNA viruses), RNA viruses (singleand double stranded RNA viruses), point mutations (any organism), singlenucleotide polymorphism (any organism), mRNA's, rTNAs, micro RNAs (fromany organism). In preferred aspects of the invention, the bioagent oranalyte is a virus or extract, a prion or a DNA or RNA molecule. Anyvirus for which a monoclonal or polyclonal antibody may be raised, forwhich a binding polypeptide is available or for which a capture DNA isavailable are particularly preferred bioagents or analytes for use inthe present invention.

Exemplary viruses which may be viral bioagents in the present inventioninclude animal, plant, fungal and bacterial viruses. Viral biogentswhich may be detected by the biosensors according to the presentinvention include those which impact animals, especially mammals, inparticular humans, domestic animals and include, for example,Papovaviruses, e.g. polyoma virus and SV40; Poxviruses, e.g. vacciniavirus and variola (smallpox); Adenoviruses, e.g., human adenovirus;Herpesviruses, e.g. Human Herpes Simplex types I and II; Parvoviruses,e.g. adeno associated virus (AAV); Reoviruses, e.g., rotavirus andreovirus of humans; Picornaviruses, e.g. poliovirus; Togaviruses,including the alpha viruses (group A), e.g. Sindbis virus and Semlikiforest virus (SFV) and the flaviviruses (group B), e.g. dengue virus,yellow fever virus and the St. Louis encephalitis virus; Retroviruses,e.g. HIV I and II, Rous sarcoma virus (RSV), and mouse leukemia viruses;Rhabdoviruses, e.g. vesicular stomatitis virus (VSV) and rabies virus;Paramyxoviruses, e.g. mumps virus, measles virus\and Sendai virus; Arenaviruses, e.g., lassa virus; Bunyaviruses, e.g., bunyawere(encephalitis); Coronaviruses, e.g. common cold, GI distress viruses,Orthomyxovirus, e.g., influenza; Caliciviruses, e.g., Norwak virus,Hepatitis E virus; Filoviruses, e.g., ebola virus and Marburg virus; andAstroviruses, e.g. astrovirus, among others.

Bioagents such as Sin Nombre virus, influenza (especially H5N1influenza), Herpes Simplex Virus (HSV1 and HSV-2), Coxsackie virus,Human immunodeficiency virus (I and II), Andes virs, Dengue virus,Papilloma, Epstein-Barr virus (mononucleosis), Variola (smallpox) andother pox viruses, West Nile virus, influenza (H5N1) find use asbioagents in the present invention.

A short list of animal viruses may be relevant bioagent targets for usein the present invention:

-   -   Reovirus    -   Rotavirus    -   Enterovirus    -   Rhinovirus    -   Hepatovirus    -   Cardiovirus    -   Aphthovirus    -   Parechovirus    -   Erbovirus    -   Kobuvirus    -   Teschovirus    -   Norwalk virus    -   Hepatitis E virus    -   Rubella virus    -   Lymphocytic choriomeningitis virus    -   HIV-1, HIV-2,    -   HTLV-I    -   Herpes Simplex Virus 1 and 2    -   Sin Nombre Virus    -   Coxsackie Virus    -   Dengue virus    -   Yellow fever virus    -   Hepatitis A virus    -   Hepatitis B virus    -   Hepatitis C virus    -   Influenzavirus A, B and C    -   Isavirus,    -   Thogotovirus    -   Measles virus    -   Mumps virus    -   Respiratory syncytial virus    -   California encephalitis virus    -   Hantavirus    -   Rabies virus    -   Ebola virus    -   Marburg virus    -   Corona virus    -   Astrovirus    -   Borna disease virus    -   Variola (smallpox virus)

Plant viruses also are relevant bioagents for use in the presentinvention. The present invention may be used to detect plant viruses,especially in agricultural applications.

Plant viruses, which may serve as bioagents (analytes) for the presentinclude the following:

Geminiviruses e.g., bigeminivirus, monogeminivirus and bybrigeminivirus;Partitiviruses, e.g., alphacryptoviruses and betacryptoviruses;Potyviruses, e.g., bymoviruses and ipomoviruses; Bromoviruses, e.g.cucumoviruses and bromoviruses; Comoviruses, e.g. fabiviruses,neopoviruses and comoviruses; Rhabodoviruses, e.g., cytorhabdoviruses,nucleorhabdoviruses; Reoviruses, e.g., oryzaviruses and phytoreoviruses;Satellite viruses, e.g., satelliviruses; Tombusviruses, e.g.,carmoviruses; Sequiviruses,e.g., sequiviruses and waikaviruses; amongnumerous others (see below).

Plant Virus Genuses, include the following:

-   -   Alfamoviruses: Bromoviridae    -   Alphacryptoviruses: Partitiviridae    -   Badnaviruses    -   Betacryptoviruses: Partitiviridae    -   Bigeminiviruses: Geminiviridae    -   Bromoviruses: Bromoviridae    -   Bymoviruses: Potyviridae    -   Capilloviruses    -   Carlaviruses    -   Carmoviruses: Tombusviridae    -   Caulimoviruses    -   Closteroviruses    -   Comoviruses: Comoviridae    -   Cucumoviruses: Bromoviridae    -   Cytorhabdoviruses: Rhabdoviridae    -   Dianthoviruses    -   Enamoviruses    -   Fabaviruses: Comoviridae    -   Fijiviruses: Reoviridae    -   Furoviruses    -   Hordeiviruses    -   Hybrigeminiviruses: Geminiviridae    -   Idaeoviruses    -   Ilarviruses: Bromoviridae    -   Ipomoviruses: Potyviridae    -   Luteoviruses    -   Machlomoviruses    -   Macluraviruses    -   Marafiviruses    -   Monogeminiviruses: Geminiviridae    -   Nanaviruses    -   Necroviruses    -   Nepoviruses: Comoviridae    -   Nucleorhabdoviruses: Rhabdoviridae    -   Oryzaviruses: Reoviridae    -   Ourmiaviruses    -   Phytoreoviruses: Reoviridae    -   Potexviruses    -   Potyviruses: Potyviridae    -   Rymoviruses: Potyviridae    -   Satellite RNAs    -   Satelliviruses    -   Sequiviruses: Sequiviridae    -   Sobemoviruses    -   Tenuiviruses    -   Tobamoviruses    -   Tobraviruses    -   Tombusviruses: Tombusviridae    -   Tospoviruses: Bunyaviridae    -   Trichoviruses    -   Tymoviruses    -   Umbraviruses    -   Unassigned potyviruses: Potyviridae    -   Unassigned rhabdoviruses: Rhabdoviridae    -   Varicosaviruses    -   Waikaviruses: Sequiviridae    -   Ungrouped viruses

A much more complete list of viral bioagents which may be analytes inthe present invention include, for example, those which are listed at:Virus Taxonomy, the Sixth Report of the International Committee onTaxonomy of Viruses (ICTV) 1995 See, the corresponding website:tulane.edu/˜dmsander/WWW/335/VirusList.html, which is referenced herein.

Prions are another class of important bioagents which may be detectedusing the invention of the present application. Exemplary prions includeScrapie (Sheep and goats), transmissible mink encephalopathy (TME),chronic wasting disease (CWD) in mule deer and elk, bovine spongiformencephalopathy (BSE) cattle, feline spongiform encephalopathy (FSE) incats, exotic ungulate encephalopathy (EUE), Kuru in humans,Creutzfeldt-Jakob disease (CJD) in humans, Fatal familial insomnia (FFI)in humans and Gerstmann-Sträussler-Scheinker syndrome (GSS) in humans.

The present invention may be used to detect bioagents in a sample, suchas viruses, prions, other microbes, biological mixtures of compositionsand components, particles and their extracts, etc. as otherwisedescribed herein. The present invention has utility in militaryapplications, especially in field applications, in commercialapplications, in agricultural applications and in research/medicalapplications (including the existence of air-born infections in thelungs of a patient). The present invention may be used to detectbioagents in the atmosphere (air), in water supplies, including standingwater, in soil samples, etc.

By way of example, the present invention is useful for detectingnumerous interactions between ligand and bioagent, including, forexample, interactions between receptor and agonist/antagonist, betweenpeptide and other peptides (peptide-peptide interaction), betweenantibody (monoclonal and polyclonal) and epitope on the bioagent(biomarkers, basic protein sequence, etc.), between polynucleotides(Watson-Crick base pairing or hybridization), as well as any otherchemical or physical interaction (e.g., Van der Waals interactions)which may specify the binding or interaction of a bioagent with theligand.

The term “tethered” is used to describe the attachment of the biologicalligand onto the surface of the biosensor. The ligands are attached tothe biosensor in a manner so that the portion of the ligand which bindsto the bioagent is exposed on the surface. The ligands may be attachedto the surface of the biosensor (preferably, a lithium tantalate chip)directly or indirectly by any means available. Ligands which are used inthe present invention are complexed (coated or tethered throughcovalent, ionic, hydrophobic or other types of bonding) with the surfaceof the biosensor. This may include employing covalent bonding betweenligand and sensor surface; e.g., the use of diazonium chemistry toattach the ligand to the sensor surface or through use of siliconechemistry. Some employ non-covalent bonding such as self-assembledmonolayers; other approaches combine both covalent binding andnon-covalent binding such as streptaviding-biotin interactions, whereinthe streptavidin molecule is non-covalently attached to the sensorsurface, and the biotin molecule is covalent bonding to the ligand andthe streptavidin and biotin molecules are covalently bound to eachother. Additionally, the attachment of ligands to sensor surface may bemade through the process of physiosorption, which relies on Van derWaals forces to make non-covalent connections to the surface.Chemisorption, which uses covalent bonds, may also be used to attach ortether ligand to sensor surface. Simple adsorption may also beapplicable to tether a ligand to the biosensor surface.

In certain applications, the surface of the piezoelectric material, alithium tantalate wafer used in the present invention, is coated with athin silicone dioxide surface or other surface which is created usingstandard methods such as sputtering techniques or more preferably,plasma enhanced chemical vapor deposition (PECVD) to produce a thinlayer (about 500 Å to about 10,000 Å or more, in certain preferredaspects, about 5000 Å) as otherwise described herein. A ligand may betethered to the silicone dioxide layer directly by covalent(chemisorption) or through non-covalent bonding (physisorption), oralternatively, the silicone dioxide layer may be further coated with ahydrophobic material (in certain embodiments, hexamethyldisilazine, orother hydrophobic material such as a liquid/semi-liquid hydrocarbon suchas petrolatum or mineral oil, including other silicone materials) toprovide a surface which is amenable to adsorption of a ligand asotherwise described herein. The hydrophobic layer on the siliconedioxide layer may be modified (covalently or non-covalently) using achemical linker such as a functional silane compound, as otherwisedescribed herein, which can covalently bond a ligand directly or bond afurther linker (a complex linker) such as a binder protein (e.g.,NeutrAvidin binding protein) which can bond certain molecules/moieties(biotin) which can be complexed with the ligand to tether the ligandindirectly to the surface of the SAW biosensor.

The term “adsorption” refers to a process that occurs when a gas orliquid solute accumulates on the surface of a solid or a liquid(adsorbent), forming a molecular or atomic film (the adsorbate).Adsorption is operative in most natural physical, biological, andchemical systems, including the present invention. Similar to surfacetension, adsorption is a consequence of surface energy. In a bulkmaterial, all the bonding requirements (be they ionic, covalent ormetallic) of the constituent atoms of the material are filled. But atomson a clean surface experience a bond deficiency, because they are notwholly surrounded by other atoms. Thus, it is energetically favourablefor them to bond with whatever happens to be available. The exact natureof the bonding depends on the details of the species involved, but theadsorbed material is generally classified as exhibiting physisorption orchemisorption.

The term “physisorption” is used to describe a physical adsorptionprocess in which there are van der Waals forces of interaction between asurface (solid) and a liquid or air layer. Physical adsorption is a typeof adsorption in which the adsorbate adheres to the surface only throughVan der Waals (weak intermolecular) interactions, which are alsoresponsible for the non-ideal behaviour of real gases. Physisorption ischaracterised by the following:

-   -   Low ambient temperature, always under the critical temperature        of the adsorbate;    -   Type of interaction: Intermolecular forces (van der Waals        forces);    -   Low enthalpy: ΔH<20 kJ/mol;    -   Adsorption takes place in multilayers;    -   Low activation energy;    -   The energy state of the adsorbate is not altered;    -   The process is reversible.

In the present invention an exemplary physisorption approach totethering ligands to the biosensor involves creates a silicon dioxide(SiO₂) layer on the biosensor wafer. This is the waveguide layer andvaries in thickness (ranges from less than about 500 Å to about 20,000 Åor more, about 1000 Å to about 10,000 Å, about 1500 Å to about 7,500 Å,about 2500 Å to about 6500 Å, about 4000 Å to 6000 Å, about 5000 Å), butis preferably about 5000 Å. Any method known in the art for depositing asilicon dioxide layer may be used, but a preferred approach utilizesplasma enhanced chemical vapor deposition (PECVD, Oerlikon Versaline,Switzerland) to provide the silicon dioxide layer. On top of the silicondioxide layer a thin layer of a non-reactive liquid silicone materialsuch as hexamethyldisilazane or other non-reactive, liquid alkylsilicone material such as an oligodialkylsiloxane, polydialkylsiloxaneor other silicone, or a other non-reactive liquid may be used may beused.

The term “chemisorption” is used to describe a type of adsorptionwhereby a molecule adheres to a surface through the formation of achemical bond, as opposed to the Van der Waals forces which causephysisorption. It is characterised by the following:

High temperatures;

Type of interaction: strong; covalent bond between adsorbate andsurface;

High enthalpy: 50 kJ/mol<ΔH<800 kJ/mol

Adsorption takes place only in a monolayer.

High activation energy

Increase in electron density in the adsorbent-adsorbate interface.

Reversible generally at high temperature.

The main way in which chemisorption is used in the present invention isthat a linker molecule which may contain at least one hydroxyl(especially, Si—OH) binding group or other binding group (to bind to thesurface of the biosensor) and at least one group which can bind an aminegroup, a carboxyl group, a hydroxyl group or a phosphate group are usedto chemically link or tether a ligand to the surface of the biosensor.Other approaches include providing a linker comprising an amine,hydroxyl, carboyxl or phosphate binding group which itself isnon-covalently physisorbed to the biosensor surface to, for example, anon-covalently bonded silicone such as hexamethyldisilazine which isitself non-covalently complexed to the SiO2 surface of the biosensor).Exemplary linkers for tethering a ligand to the biosensor include, forexample, silanes containing reactive functional groups (“functionalsilane”), for example, 3-aminopropyltrimethoxysilane (amino groupbinding) and 3-glycydoxylpropyltriethoxysilane (carboxyl group binding),among others.

Several preferred approaches to tethering ligands may be taken in thepresent invention. These approaches may involve exclusivelyphysisorption methods, chemisorption methods or combinations ofphysisorption and chemisorption methods. In a first physisorptionmethod, a layer of silicon dioxide is produced on the surface of thebiosensor using plasma enhanced chemical vapor deposition (PECVD)technology. Once the silicon dioxide layer is in place on the biosensor,a thin layer of hexamethyldisilazine (HMDS) or other inert silane isplaced on the silicone dioxide layer. An antibody or peptide may bebonded (physisorbed) to the biosensor through the hexamethyldisilizanelayer.

Alternatively, an antibody, peptide, fatty acid or carbohydrate may bebonded to a chemical linker (e.g. a functional silane) which is itselfbonded (physisorbed) to a layer of hexamethyldisilazine (HMDS) andsilicon dioxide to provide a tethered ligand on the biosensor of thepresent invention. This method combines both physisorption andchemisorption methods.

Still another approach for tethering a ligand to the biosensor of thepresent invention involves utilizing a linker protein, such asNeutrAvidin Binding Protein which itself binds to a biotinylated ligand,such as a peptide, amino acid or DNA molecule. In this aspect, thebiosensor is first provided with a silicon dioxide layer to which islayered (physisorbed) a non-reactive silicone liquid, e.g.,hexamethyldisilizane (HMDS). A thin layer of a functional silane, forexample, 3-aminopropyltrimethoxysilane (amino group binding) and3-glycydoxylpropyltriethoxysilane (carboxyl group binding), among othersmay be physisorbed to the hexamethylsilazine layer and the functionalsilane may be used to covalently link a binding protein (e.g.NeutrAvidin Binding Protein) to the biosensor. The NeutrAvidin BindingProtein may then be used to bind any biotinylated ligand. Additionalapproaches, or variations on the above approaches to tether virtuallyany ligand to the biosensor of the present invention are well within theordinary skill of the routineer.

The present invention is based on a biosensor apparatus featuringsurface chemistries that are capable of transducing shear horizontalsurface acoustic waves (SH-SAW) generated by piezoelectric materialssuch as lithium tantalate (LiTaO₃) when connected to an electricalcircuit at defined frequencies. This technology is manufactured ontolithium tantalate wafers (or wafers from another piezoelectric material)that can be sectioned into functional units (“chips”). These units canbe applied to a SAW detection board featuring a fluidic housing(described below) and a connection of the chip via aluminum delay linesto an output interface with a laptop computer for measurements of SAW.FIGS. 1B and 1C (attached) represent the functional unit. FIG. 1D is ablock diagram of functional components of the ZAW measurement system.

As depicted in FIG. 1D, the components of the SH-SAW detector includethree transducer elements 102, 104, 106 in the form of IDT electrodesdisposed in a linear array on a substrate 108. Electrodes 102, 104, 106are connected at inputs to a power source 110 via a power splitter 112.A reference oscillator 114 is provided, which may be used in part togenerate the SH-SAW waves in the fluidic housing. As shown in FIG. 1B,reference oscillator 114 is disposed on the same substrate or chip asIDT electrodes 102, 104, 106. IDT electrodes 102, 104, 106 are eachconnected on an output side via a respective delay line 116, 118, 120 toa respective phase difference detector 124, 126, 128 exemplarily in theform of an IQ demodulator. Phase difference detectors 122, 124, 126 alsoreceive input from oscillator 114 via a reference line 122. Phasedifference detectors 122, 124, 126 are connected to a computer 130 viaanalog-to-digital converters 132. Computer 128 may be programmed, intercilia, to issue an alert signal via an output transmitter 134 upondetecting a disturbance in a standing wave indicating the presence of atarget pathogen or toxic agent.

In FIG. 1B, the sensor wafer or substrate 108 is shown in scale comparedto a dime coin (10 US cents); the four aluminum delay lines are visible;one line 122 serves as the reference and three others 116, 118, 120(FIG. 1D) as the test delay lines. In FIG. 1C, the SAW detection board(thin arrow) with the fluidic housing (thick arrow) and the outputinterface device (arrow head) to a laptop computer is shown.

As described above, the surface of the sensor wafer can be complexedwith ligands that are specific for any biological substance (forexample, but not exclusively, a protein), free or as part of a largercomplex (for example a virus, a spore, or a cell) for its detection whenthis substance, added in solution to the fluidic housing (the bioagentpasses through small holes in the housing, which is permeable to thebioagent), is specifically recognized by its specific ligand on thesurface of the sensor (see Claims below). Specific recognition (binding)results in a characteristic and quantifiable change of frequency that iscaptured using software applicable for such a purpose. The presentinvention is directed to the detection of biological entities, such asviruses and cells, of both prokaryotic and eukaryotic nature, as well asprions and molecules using ligands specific for surface molecules ofthese entities. Such ligands can be for example antibodies or peptides.FIG. 1A shows one such example, in which a biotinylated antibody that isable to specifically recognize a virus is coupled to the LiTaO₃ surfaceof a SAW biosensor that has been coated with NeutrAvidin Biotin BindingProtein.

Preferred Embodiment

The following describes a preferred embodiment. The present invention isdirected to the detection of viruses and other biological and chemicalagents by SAW. In this embodiment, a Coxsackie virus, a member of theenterovirus family, which also includes polioviruses and hepatitis Avirus (Palacios and Oberste, 2005) was the bioagent. Coxsackie virusinfections are usually not of major health concern and possible fevers,headaches, and muscle aches typically self-dissolve after several days.Occasionally however, Coxsackie virus can cause serious infections, suchas hand, foot and mouth disease, viral meningitis, encephalitis,myocarditis, and hepatitis, especially in newborns (Frydenberg andStarr, 2003; Tam, 2006). The primary objective is the Sin Nombre virus(SNV), a hantavirus member of the Bunyaviridae family of RNA viruses anda category A agent as defined by the National Institute of Allergy andInfectious Diseases (NIAID; website niaid.nih.gov) and other category Abioagents. SNV is transmitted by its reservoir host Peromyscusmaniculatus, the deer mouse. Transmission happens by inhalation ofaerosolized feces, urine, or saliva from the infected mice (Jay et al.,1997). The illness that ensues, hantavirus cardiopulmonary syndrome(HCPS), is characterized initially by mild flu-like symptoms, followedby rapid progression to respiratory distress, and can be fatal (Hjelle,2002; Mertz et al., 2006). There is no established therapeutic regimenand treatment is only supportive. Preventive methods include attempts tominimize contact with the rodents since elimination of the virus is notrealistic. Although human infections with hantaviruses are on the rareside, these agents have been classified by the Center of Disease Control(CDC) as potential agents for biologic terrorism due to their relativeease of production, the high susceptibility of large populations, andthe limited treatment and vaccination strategies (Bronze et al., 2002).

In the present invention, the development and preliminary use of anSH-SAW biosensor able to detect category A viral agents operating at aninput frequency of 325 MHz was a principal objective. Input frequenciesmay vary over a range of about 275 to 400 MHz, with 315-330 and 325 MHzbeing particularly useful. The present invention shows that thebiosensor device of the present invention is sensitive and highlyselective for its specific target. We also provide data in support ofthe biosensor's high reproducibility and its use in a real worldscenario mimicking the exposure of an urban population to a viral agent,such as for example through the water system. The present inventionindicates that this detection platform is highly versatile and robustand has significant medical or defense use.

2. Materials and Methods 2.1. Fabrication of the 325 MHz SH-SAW Sensor

Wafer preparation and lithographic deposition of IDT layer: The SH-SAWdevice was fabricated using metal evaporation and lift-off plasmaenhanced chemical vapor deposition (PECVD), and reactive ion etching(RIE) techniques on a 36° y-cut, x-propagating lithium tantalate(LiTaO₃) crystal wafer of 510 μm thickness and 100 mm diameter. Thewafer was cleaned in a barrel asher (PVA Tepla, Asslar, Germany) for 5minutes, 600 Watts power and 900 mTorr of O₂, followed by dipping in 1vol % hydrofluoric acid (HF) for 3 minutes, and rinsing in a cascadebath until the resistivity of the water was greater than 12 MOhm-cmfollowed by drying under N₂. The wafer layout was designed to includefour IDT patterns with sets of transducers and delay lines. AZ2020 (AZElectronic Materials, Branchburg, N.J., USA) negative-tone photoresist(PR) was applied using a spin coater with a Gyrset lid (Karl-Suss,Waterbury Center, Vt., USA) at 1300 and 3000 rpm/sec for 30 seconds eachto achieve a thickness of 2.0 μn. The wafer was baked on a hotplate for60 seconds at 110° C. and cooled to room temperature on a metal surface.Because of the pyroelectric nature of LiTaO₃ it was necessary to removeresidual electric charge by dipping the wafer in de-ionized water anddrying under N₂. The PR was exposed to i-line UV light at 365 nm for 6seconds at 14 mW/cm². The wafer was re-baked on a hotplate at 110° C.for 60 seconds followed by a 1 minute developing time in a 300 MIFdeveloper (AZ Electronic Materials) and rinsing in de-ionized water. Thewafer was metallized with 5000 Å aluminum using an electron-beamevaporator (Temescal, Wilmington, Mass., USA). The deposition rate was 3Å/sec for 500 Å and then 5 Å/sec for 4500 Å. The wafer was placed in anacetone bath to lift off the PR and excess aluminum. An acetone spraywas used to remove the PR between the IDT fingers, followed by rinses inmethanol, isopropyl alcohol, and de-ionized water. This procedure wasrepeated for the metallization of the ground plane, buss lines, andcontact pads.

Deposition of the waveguide layer and final preparation: A 5000 Åsilicon dioxide (SiO₂) film was deposited onto the entire wafer usingPECVD (Oerlikon Versaline, Pfaeffikon, Switzerland) at 150° C. for 410seconds. The oxide was coated with hexamethyldisilazane (HMDS) in avacuum oven at 100° C. for 30 minutes. AZ4330 positive-tone PR (AZElectronic Materials) was spin coated onto the wafer at 2000 rpm and3000 rpm/sec for 30 seconds each. The wafer was baked on a hotplate at90° C. for 90 seconds, exposed for 48 seconds at 20 mW/cm² (at 400 nm),and developed in a 300 MIF developer for 3 minutes. A photoresist maskwas used to “open” the SiO₂ and expose the electrical contact pads. TheSiO₂ was etched by RIE (Oerlikon Versaline) for 1500 seconds (p=40 mT,CHF3=45 sccm, O2=5 sccm, P=125 watts, DC bias=712 volts). The wafer wasdiced using a resinoid dicing blade. The PR was removed from theindividual die by rinsing in acetone, methanol, and isopropanol.

2.2. Virus Production and Preparation

Viral work was conducted in Biosafety Level 2 (Coxsackie virus B4) orLevel 3 (SNV) facilities at the University of New Mexico School ofMedicine. The JVB strain of Coxsackie virus B4 was purchased from ATCC(Manassas, Va., USA). Buffalo Green Monkey Kidney (BGMK) cells werepurchased from Diagnostic Hybrids, Inc. (Athens, Ohio, USA). BGMK cellswere grown to confluency in DMEM, 2 mM L-glutamine, 10 mM HEPES, 26.8 mMsodium bicarbonate, and 10% fetal bovine serum. After removal of culturemedia, BGMK cells were inoculated with Coxsackie virus B4 for 1 hour atroom temperature and grown in a serum free media, Opti PRO SFM (GIBCO,Grand Island, N.Y., USA) supplemented with 4 mM L-glutamine. Uponreaching 100% cytopathic effect (CPE), the media was centrifuged in asealed rotor for 5 minutes at 3000 rpm and the supernatant was stored at−20° C. The viral titer was determined by serial dilution and growth inBGMK cells. The virus was inactivated with 2.0 Mrads of gamma radiationusing a Gammacell 40 (Atomic Energy of Canada, Ltd., Kanata, Ontario,Canada) and concentrated by lyophilization and resuspension in distilledwater.

SNV particles were purified from the supernatant of VeroE6 monkey kidneycells (ATCC) infected with SNV at a multiplicity of infection of 0.1 for1 hour. After 9 days the media was subjected to ultracentrifugationusing Optiprep density gradients in phosphate buffered saline (PBS)(Axis-Shield, Norton, Mass.) and the isolated virus was inactivated byUV. The amount of SNV particles was quantified by determining the SNVnucleocapsid (SNV-N) protein concentration (not shown). SNV particlenumber was calculated based on the following parameters pertaining tothe SNV-N protein: SNV-N has a mass of 50 kD; thus 1 mol(6.02×10²³)=50,000 grams; thus one SNV-N protein=8.3×10⁻¹¹ nanograms.There are ˜10⁵ SNV-N proteins per SNV particle; thus 8.3×10⁻⁶ nanogramsof SNV-N=one SNV particle. Using these calculations, 1 nanogram of SNV-Nprotein corresponds to ˜120,000 SNV particles. Purified Herpes Simplexvirus type 1 (HSV-1) was a kind gift of Dr. Stephen Young at TriCoreLaboratories (Albuquerque, N. Mex., USA).

2.3. Preparation of Antibodies and Adsorption to the SAW Biosensor

The IgG2a isotype monoclonal antibody (mAb) directed against the JVBstrain of Coxsackie virus B4 was produced by the 204-4 hybridoma cellline (ATCC). The mAb was labeled with biotin using the EZ-Link NHS-PEOSolid Phase Biotinylation Kit and Spin Column featuring the SwellGelDisk technology (Pierce, Rockford Ill., USA). The mAb was added to thegel at 2 mg/ml for 10 minutes followed by incubation in NHS-PEO4-Biotinfor 30 minutes at room temperature and column spinning. The biotinlabeled antibody was eluted from the column with 0.2 M Imidazole in PBS.Single chain Fv antibodies (scFv) directed against SNV have beenpreviously selected from a phage antibody library, and are specific forthe SNV-G1 glycoprotein exerting no cross-reactivity with glycoproteinsfrom other hantaviruses (Velappan et al., 2007). These scFv antibodieswere left unmodified and directly adsorbed to the biosensor.

The SAW devices were cleaned in acetone, methanol, and isopropanol, thentreated by ultrasonication in 95% ethanol, rinsed in distilled water,followed by exposure to UV-ozone for 10 minutes in a UVOCS UV-Ozonecleaner (UVOCS Inc., Montgomeryville, Pa., USA). For the antibodydirected against the Coxsackie virus B4, the oxide layer of the testdelay lines of the SAW device were coated in a non-covalentphysisorption process with 0.25 mg/ml of NeutrAvidin Biotin BindingProtein (Pierce) in PBS for 30 minutes at room temperature. The devicewas washed 3 times with PBS and 1 time with distilled water, and driedusing nitrogen. The biotin labeled 204-4 mAb was adsorbed to theNeutrAvidin Biotin Binding Protein at 0.25 mg/ml in PBS for 30 minutesat room temperature. For the detection of SNV, anti-SNV-G1 scFv at 0.25mg/ml was directly adsorbed to the SAW device for 30 minutes at roomtemperature. After coating with the antibodies, the biosensor deviceswere washed 3 times with PBS and 1 time with distilled water, followedby nitrogen drying.

2.4. SAW detection of Coxsackie and SNV

For display and acquisition of the digitized voltage data from thesensor platform, a custom LabVIEW (National Instruments; website ni.com)program was developed. The data from each of the delay lines wereacquired simultaneously and the voltage information was converted backto the phase. Data from the reference line were subtracted from the datafrom the test lines for each time point measured and continuously savedto disk in spreadsheet format. The data for the quantitativemeasurements of viral detection presented in this study refer to thephase differential mass shift (Δφ) and are displayed on a graph as afunction of time of acquisition. Multiple data points per time arerecorded during this process. The SAW device was assembled in a staticcell with a 325 MHz input frequency from a power supply. The chamber(fluidic housing) was covered with 0.4 ml of PBS or medium and the phasedifferential was stabilized for at least 100 seconds. 0.1 ml of solutionwas removed and replaced with 0.1 ml of virus containing solution. Viruswas typically detected within ˜15 seconds as observed by an elevation ofthe signal Δφ (in degrees on the y-axis) along the time plot (x-axis).The detection run was allowed to reach stabilization of the phasedifferential, typically after ˜2 minutes. The virus containing solutioncan be removed and the run can be continued with 0.4 ml of PBS untilstabilization. Δφ can either be measured at stabilization after agentinjection or after removal of the agent and re-stabilization in buffer.A detailed description of the former process is given in the legend forFIG. 2.

2.5. Statistics

All phase differential mass shift (Δφ) data points were generated in atleast duplicate and sometimes up to five tests. Data is presented asaverage±standard errors (SE). The statistical difference between groupsof average measurements of phase differential mass shifts Δφ weredetermined using the students t-test. P<0.05 was considered to bestatistically significant.

3. Results

3.1. Initial testing of the SAW detector

The major objective of this study was to couple the characteristics ofSAW with the selectivity of antibodies to detect viruses of high medicaland bio-warfare importance. FIG. 1A depicts the overall schematicconcept of our device. FIG. 1B shows the miniature size of the wafersensors, i.e. approximately 15 by 20 mm, featuring 4 delay lines, ofwhich one is used as the reference line, while the others are used astest lines. FIG. 1C shows the SAW detection board with the fluidichousing (containing the wafer) and the output interface device to alaptop computer. The sealed fluidic housing connected the IDTs to theelectrical input/output system of the static cell; a power supplyprovided an input frequency of 325 MHz. The phase differential massshift Δφ (expressed in degrees) was captured on the laptop computerusing a custom LabVIEW program.

Viral detection was performed by immobilizing antibodies onto thesensor. The test delay lines were either directly coated with unlabeledantibodies (for SNV) or with NeutrAvidin Biotin Binding Protein followedby botinylated antibodies (for Coxsackie virus B4). FIG. 2 represents atypical phase differential plot generated by exposing the sensor to1.8×10⁴ SNV particles per μl. The plot shows the PBS buffer calibratedsurface at ˜50 degrees, injection of the virus containing solution at˜280 seconds (measured on the x-axis), and maximal signal at ˜420seconds (˜2 minutes after agent injection). Detection was evident at ˜15seconds after addition of the agent and resulted in an overall Δφ of ˜5degrees (measured on the y-axis). Thus, Δφ can be measured shortly afteragent injection and the overall process can be performed within minutes.All subsequent data for the quantitative measurements of viral detectionrefer to the change in Δφ as described in FIG. 2.

3.2. Quantitative and Selective Detection of Coxsackie B4 and Sin NombreVirus (SNV)

The SAW biosensor was used in a series of experiments for the detectionof Coxsackie virus B4 particles (FIG. 3). Increasing concentrations ofviral particles ranging from 9×10⁵ to 3.6×10⁶ viruses per μl wereanalyzed as described in FIG. 2. Δφ was recorded for each concentrationand resulted in a dose-dependent increase with Δφ values ranging from0.95±0.54 to 6.99±0.96 (FIG. 3; data set to the right). There was alinear relationship between viral load and Δφ for this range of viralparticles with a correlation coefficient R² of 0.99. To determine theselectivity of the biosensor for its target in the presence of aconfounding agent, the experiments were repeated using admixtures ofCoxsackie B4 and HSV-1 viruses. HSV-1 was spiked into the Coxsackievirus preparations at equal final concentrations as for Coxsackie B4 andthe previous experiments were repeated. HSV-1 did not affect theselectivity for Coxsackie B4, yielding similar Δφ values ranging from1.09±0.66 to 7.24±0.36. The relationship between viral load and Δφremained linear with a correlation coefficient R² of 0.98. In thesespiking experiments, where HSV-1 was used as a confounding agent, the Δφfor HSV-1 at a concentration of 3.6×10⁶/μl was 0.64±0.33, indicatingthat the biosensor did not detect HSV-1.

Next, we used the SAW biosensor to detect an agent of high medicalconcern, i.e. SNV, a member of the hantavirus genus of the familyBunyaviridae and an NIH-designated bioagent of category A (Bronze etal., 2002). Concentrations of 1.8×10¹ to 1.8×10⁴ viral particles per μlresulted in Δφ of 0.63±0.49 to 4.85±0.77 (FIG. 3; data set to the left).The measured values displayed variation with some overlap as shown bytheir standard deviations. This can be attributed mainly to factorsrelated to microfabrication reproducibility for the current sensorsystem. Our studies indicate that sensitivity is a function of theabsolute thickness of the silicon dioxide waveguide. We have determinedthat thickness variations of 5% lead to a ˜8% alterations in detectionsensitivities (data not shown). However, our calculated waveguidevariations for the current sensors are approximately 1-2%, which isunlikely to contribute to the observed experimental variation. Inaddition, variations in the lithographic process which guides thegeometry of the IDTs on our sensors can influence their sensitivity. Weare currently gathering data to quantify this potential problem and maychange the process from contact to projection lithography. Nevertheless,the data means resulted in a linear relationship between viral load andΔφ for this range of viral particles with a correlation coefficient R²of 0.95. Similar to the experiments with Coxsackie virus, theselectivity of the SAW biosensor for SNV was not diminished by thepresence of confounding HSV-1 at equal concentrations. The correspondingΔφ ranged from 0.78±0.30 to 4.83±1.36 and the relationship between viralload and Δφ remained linear with a correlation coefficient R² of 0.97.For this set of experiments, the biosensor was marginally sensitive toHSV-1 alone, resulting in Δφ values of 0.56±0.27 and of 1.51±0.40 forviral concentrations of 1.8×10³/μl and 1.8×10⁴/μl, respectively.

Based on the lowest concentrations tested for both viral agents, whichin both cases yielded Δφ above values obtained with HSV-1, thesensitivity for the antibody directed against SNV was approximately50×10⁴-fold greater compared to the sensitivity of the antibody directedagainst Coxsackie B4 virus. The slopes for the detection of Coxsackievirus and SNV were markedly different (FIG. 3). This could be due tomultiple factors including the deposition efficiency and correspondingsite density of the capturing agents coated on the biosensor surface, inthis case the anti-Coxsackie B4 and anti-SNV antibodies, whichdetermines the number of binding sites available to specific viralligands, in this case the Coxsackie B4 JVB epitope and the SNV-G1glycoprotein. Similarly, differences in sensitivity can be explained byits dependence on the affinity between an antibody and its correspondingantigen. This leaves room for the development of better immunologicaltools with higher sensitivities. Accordingly, we are currently employingphage display to identify high affinity protein-peptide interactions toincrease the sensitivity for viral particles.

3.3. Virus Detection in Complex Solutions

The SAW biosensor was applied to a real world scenario which could mimicthe exposure of an urban population to a viral agent, such as forexample through the water system. In these proof-of-principleexperiments, SNV particles were spiked into 0.4 ml of either the sewageeffluent water of the City of Albuquerque N. Mex., water taken from theRio Grande River in Albuquerque, or distilled water or PBS as controlsat a final concentration of 1.8×10⁴ virus/μl. As shown in FIG. 4, all ofthe control solutions lacking viral particles showed similar backgroundlevels of Δφ in the order of 0.1. The SAW biosensor showed the highestsensitivity when the viral particles were spiked into distilled water,displaying a Δφ of 8.21±0.11 degrees (˜82× over background). The Δφ forvirus spiked into PBS, Rio Grande River water, and sewage effluent waterwere similar, i.e. 4.85±0.77, 5.36±0.93, and 4.22±0.84, respectively,corresponding to ˜48×, 54×, and 42× fold differences over background,respectively. The Δφ values of all virus spiked solutions weresignificantly (p<0.05) higher than their background solutions withoutvirus, as determined by simple students t-test. In addition, theseexperiments in complex solutions collectively indicate a highre-usability of the sensor resulting in reproducible data. The data inFIG. 4 represent duplicate experiments using regenerated sensors aftertreatment with organic solvents, ultrasound, and UV-ozone. Nevertheless,multiple antibody re-coating and re-testing procedures led to Δφmeasurements for the specific target within standard errors of ˜13% ofthe mean.

4. Discussion

Antibody based detection by SAW has previously been reported forcellular microorganisms, such as bacteria. We recently reported on a 103MHz operated SAW sensor to detect bacterial spores of Bacillusthuringiensis, a pathogen simulant for Bacillus anthracis, at or belowinhalational infectious levels (Branch and Brozik, 2004). In that studywe developed suitable chemistries to orient the antibodies on the sensorusing protein G on two different waveguide materials, i.e. polyimide andpolystyrene. We found that polyimide had a lower mass detection limitleading to highly selective bacterial detection as shown by the absenceof binding to the control agent Bacillus subtilis. Similarly, Berkenpasand colleagues recently described a SAW biosensor that was coated with apolyclonal antibody specific for the toxigenic Escherichia coli O157:H7bacterium which causes hemorrhagic colitis and hemolytic uremic syndrome(Berkenpas et al., 2006). These authors reported Δφ responses ofapproximately 14 degrees representing a 7-fold difference overbackground control antibodies against trinitrophenyl (TNP) hapten.

In contrast, detection of viral and other agents by antibody coupled SAWis largely absent from the literature. In one of the few publishedstudies Tamarin and colleagues developed a SAW sensor similar to the onepresented here, and used it to study physical parameters of specificbinding between antibodies and M13 bacteriophages (Tamarin et al.,2003). In addition, Tassew and Thompson reported on the detection of thehuman immunodeficiency virus (HIV) type 1 Tat protein using immobilizedtrans-activation-responsive RNA elements and various Tat peptidefragments (Tassew and Thompson, 2003). Thus, our study represents acontribution to the field of specific detection of viral agents by theSAW technology based on protein interactions (antibodies).

Collectively, our experiments using Coxsackie B4 and SNV indicate a highsensitivity and selectivity of the SAW detector for its targets.Importantly, selectivity was not compromised by the presence ofconfounding and related factors, such as other viruses, indicating thepotential use of this device for the specific detection in complexsolutions. In a previous study we have used quantitative (real time)reverse transcriptase polymerase chain reaction (RT-PCR) specific forthe S segment of SNV RNA to determine viral copy number in bodilyfluids, such as blood plasma and tracheal aspirates (Xiao et al., 2006).Assay sensitivity was ˜5000 RNA copies (corresponding to viralparticles) per ml, but the material to be tested had to be processed.Pre-processing of analyte is also necessary for other establishedtechniques, including enzyme linked immunosorbent assay (ELISA) andsurface plasmon resonance (SPR). Furthermore, SPR technology is stillrelatively costly and not amenable to a portable format. In contrast,our SAW platform allows detection of viral bioagents withoutpre-processing of the analyte, and can be developed into a portablefield application. The SAW biosensor was able to detect at least 7000SNV particles. Although the minimal infectious dose of SNV necessary toestablish a successful human infection remains unknown, this number issubstantially lower than the dosage found in hantavirus infected humanssuffering from HCPS (Xiao et al., 2006).

An important and novel finding of this study is the detector'sapplication to a real world scenario using water obtained from thesewage system and from a river of an urban area. This is a real worldpossibility for planned or accidental bioagent distribution and can leadto mass exposure within the population, which in turn can lead to anextensive health crisis (Bronze et al., 2002; www.cdc.gov). Naturallyoccurring water bodies, such as rivers are admittedly very complexsolutions containing perhaps millions of different organic and inorganicmolecules. It is thus noteworthy that our antibody-coated biosensor wasable with high selectivity to detect the correct agent against thiscomplex background. While the likelihood of detection in a large body ofwater could be increased by concentration strategies, such a procedurewould compromise two important strengths and advantages of the currentsystem, i.e. the hand-held, portable nature, and the absence of analytepre-processing. In addition, it remains to be shown whether aflow-through set-up that continuously monitors dilute samples vs. thestatic set-up reported here would increase the representative power ofthe analyzed sample. In a static setting changes in phase differentialdue to the added agent and buffer are accumulating, while in aflow-through setting the sensor would return back to the original phasedifferential of the carrier buffer, as the sample passes through thesensor. Comparisons between these approaches are currently underinvestigation in our laboratory.

Together, these data, and the results which are presented in Table 1,below, indicate that the sensor of the present invention may be usedunder field conditions and warrant further efforts into the developmentof inexpensive portable devices for field operation. This includes ahand-held battery operated and self-contained version of the presentinvention.

5. Further Examples

The following ligands (presented in Table 1) have been successfullyadsorbed to the SAW biosensor and the corresponding analysis using thebiosensor/SAW technology as described above have been successfullyimplemented. The analytes (Table 1) were detected selectively, partiallyin the presence of confounding agents (agents which are provided to makeit more difficult to evidence binding and analysis by the presentinvention).

Importantly, the analytes were rapidly detected in real-time and incomplete absence of any pre-processing steps.

Methods of Tethering Ligand to SAW Biosensor

The surface of the SAW biosensor can be prepared for ligand bindingusing the following different procedures/methods which are applicable todifferent ligands as listed in the Table. The sequential steps aregenerally described for each Method (also, see above), and the lettersare listed in the third column (under “Absorption Mode”) for thecorresponding ligands. The following coats are deposited onto thelithium tantalate (LiTaO₃) surface.

Method A

1. A layer of silicone dioxide is etched/deposited onto the surface ofthe lithium tantalate surface. This is the waveguide layer and is ˜5000Å thick.2. A thin layer of hexamethyldisilazane (HMDS) is deposited onto thesurface of the silicon dioxide.3. Ligand is deposited onto the HMDS layer non-covalently.

Method B

1. A layer of silicone dioxide is etched/deposited onto the surface ofthe lithium tantalate surface. This is the waveguide layer and is ˜5000Å thick.2. A thin layer of hexamethyldisilazane (HMDS) is deposited onto thesurface of the silicon dioxide.3. A thin layer of 3-aminopropyltrimethoxy silane (amino group binding)or 3-cycydoxylpropyltriethoxy silane (carboxyl group binding) as afunctional silane is deposited onto the HDMS layer.4. Ligand is bound to the functional group (amine or carboxyl group) ofthe silane covalently.

Method C

1. A layer of silicone dioxide is etched/deposited onto the surface ofthe lithium tantalate surface. This is the waveguide layer and is ˜5000Å thick.2. A thin layer of hexamethyldisilazane (HMDS) is deposited onto thesurface of the silicon dioxide.3. A thin layer of 3-aminopropyltrimethoxy silane (amino group binding)or 3-cycydoxylpropyltriethoxy silane (carboxyl group binding) as afunctional silane is deposited onto the HDMS layer.4. A thin layer of NeutrAvidin Binding Protein (0.25 mg/ml-range of 0.05mg/ml to 1 mg/ml, about 0.1 mg/ml to about 0.75 mg/ml, about) inphosphate buffered saline is layered onto the layer of functionalsilane. A covalent linkage between the protein and functional silaneresults.5. Ligand is bound to the functional group (amine or carboxyl group) ofthe silane covalently.

TABLE 1 Analyte (recognized Adsorption Ligand by ligand) Mode AntibodiesAnti-Coxsackie virus monoclonal antibody (mouse IgG2a) CoxsackieMethods A, B, virus B4 Method C (if ligand (JVB strain) biotinylated)Anti-Sin Nombre virus (SNV) G1 glycoprotein (single Sin NombreMethods A, B, chain Fv, scFv from phage display) virus (SNV)Method C (if ligand biotinylated)Anti-Herpes Simplex virus 1 (HSV-1) antibody Herpes Methods A, B,(polyclonal) Simplex virus Method C (if ligand 1 (HSV-1) biotinylated)Anti-avβ3 integrin antibody avβ3 integrin  Methods A, B,Method C (if ligand biotinylated)Anti-Interleukin-12 (IL-12) monoclonal antibody (mouse HumanMethods A, B, IgG1) Interleukin- Method C (if ligand 12 (IL-12)biotinylated) Anti-Interleukin-6 (IL-6) polyclonal antibody (goat IgG)Human Methods A, B, Interleukin-6 Method C (if ligand (IL-6)biotinylated) Peptides18 different peptides (cyclic, 9 amino acids; from phage Sin NombreMethods A, B, display) virus (SNV) Method C (if ligand18 different peptides (cyclic, 9 amino acids; from phage biotinylated)display) 1) JC-1 = CLVRNLAWC SEQ ID No 1 2) JC-2 = CSASTESLC SEQ ID No 23) JC-3 = CQTTNWNTC SEQ ID No 3 4) JC-4 = CKSFTTTRC SEQ ID No 45) JC-5 = CAEPNSHRC SEQ ID No 5 6) G1-0 = CKQTTNRNC SEQ ID No 67) G1-1 = CQATTARNC SEQ ID No 7 8) CSAGAPEFC SEQ ID No 89) CTQSGLLSC SEQ ID No 9 10) CKSFTTTRC SEQ ID No 1011) CTYPYPKFC SEQ ID No 11 12) CKSTFSPNC SEQ ID No 1213) CTSAAVHMC SEQ ID 13 14) CQPHLPWHC SEQ ID 1415) CQWPGQSGC SEQ ID No 15 16) CNSSSPTAC SEQ ID No 1617) CHQLMQNLC SEQ ID No 17 18) CIQSGLLS SEQ ID No 1810 different peptides (cyclic, 9 amino acids; from   Andes virusMethods A, B, phage display) Method C (if ligand1) C5-1 CPKLHPGGC SEQ ID No 19 biotinylated)2) C5-2 CPMSQNPTC SEQ ID No 20 3) C5-3 CTVGPTRSC SEQ ID No 214) B9-1 CPSNVNNIC SEQ ID No 22 5) B9-2 CMQSAAAHC SEQ ID No 236) B9-3 CNSHSPVHC SEQ ID No 24 7) B9-4 CKSLGSSQC SEQ ID No 258) B9-5 CPAASHPRC SEQ ID No 26 9) B9-6 CEKLHTASC SEQ ID No 2710) B9-7 CSLHSHKGC SEQ ID No 281 peptide (linear, 16 amino acids; from phage display) Dengue virusMethods A, B, DTRACDVIALLCHLNT SEQ ID No 29 Method C (if ligandbiotinylated) Nucleic Acids (a) SNV-N (nucleocapsin protein) Capture DNASin Nombre Method C 1) CCCTAGAGATGCTGCATTGGCAACTAA SEQ ID No 30virus (SNV) 2) TTACAGGTTGATGAGTCAAAAGTTAGT SEQ ID NO 31 RNA3) TTACAGGTTGATGAGTCAAAAGTTAGTGATATTGAGGACC SEQ ID NO 32(b) SNV-M (glycoprotein) Capture DNA1) CCTGATCAAAATGGACAAGGTTTAATGAGAATAGCTGGGC SEQ ID No 332) TTTCATGCTCACATTATTCTACAGAAT SEQ ID No 343) TTTCATGCTCACATTATTCTACAGAATCAAAATTCAAAGT SEQ ID No 35Biotinylated BRCA1 capture DNA (single-stranded; 30 BRCA1 DNA Method Cbases) (single- 5′-CCTGGATAATGGGTTTATGAAAAACACTTT SEQ ID No 36stranded; 60 bases) Physisorption = Direct adsorption on silicon dioxideSilane = 3-aminopropyltrimethoxy silane NAB = NeutrAvidin BindingProtein

6. Conclusions

Detection of biological agents and their products in the context ofbio-warfare, human health and agricultural production are importanttasks with a current sense of urgency. Applications include thedetection of agents and their products in the environment after plannedor accidental distribution into the soil or water bodies, such asswimming pools, rivers, aquifers, and the sewage system, especially inhighly urbanized areas. Another application is the detection of agentsand markers thereof in human fluids and tissues for the correctdiagnosis and treatment stratification of patients. Towards this end, wehave developed a SAW biosensor that is capable of rapidly detectingviral agents with high selectivity and sensitivity for at least twodifferent targets, i.e. Coxsackie B4 virus and the category A bioagentSNV. Our detector combines the sensitivity of surface acoustics with theselectivity of antibody-antigen recognition and offers a highlyversatile platform for the detection of other viral agents and theirproducts. Further, the results obtained in this study emphasize thepossibility for future, more refined developments and applications ofportable SAW based technology in the fields of viral medicine andenvironmental surveillance.

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1. A ligand based biosensor comprising a biological ligand complexed,directly or indirectly to a surface of a piezoelectric material, saidpiezoelectric material being operably connected to an electric circuitcapable of producing surface acoustic waves on the surface of thepiezoelectric material to detect the binding of a bioagent or analyte tosaid ligand based biosensor.
 2. The biosensor according to claim 1wherein said biological ligand is a polypeptide, a DNA or RNA molecule,a lipid or carbohydrate.
 3. The biosensor according to claim 1 whereinsaid piezoelectric material is lithium tantalate ((LiTaO₃).
 4. Thebiosensor according to claim 1 wherein said ligand is an antibody. 5.The biosensor according to claim 4 wherein said antibody is a monoclonalantibody.
 6. The biosensor according to claim 4 wherein said antibody isa polyclonal antibody.
 7. The biosensor according to claim 1 whereinsaid ligand is a polypeptide.
 8. The biosensor according to claim 1wherein said ligand is a polynucleotide.
 9. The biosensor according toclaim 8 wherein said ligand is a DNA molecule.
 10. The biosensoraccording to claim 8 wherein said ligand is a RNA molecule.
 11. Thebiosensor according to claim 1 wherein said ligand is a lipid or acarbohydrate.
 12. The biosensor according to claim 1 wherein said ligandis covalently linked to said piezoelectric material.
 13. The biosensoraccording to claim 1 wherein said ligand is non-covalently linked tosaid piezoelectric material.
 14. The biosensor according to claim 1wherein said bioagent or analyte is selected from the group consistingof viruses and their extracts, prions and their extracts, eucaryoticcells, fragments and extracts, prokaryotic cells, fragments andextracts, prokaryotic spores, fragments and extracts, protein andpeptide markers, serum proteins, isolated proteins, synthethicchemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts,including mitochondria and other organelles, prokaryotic membranes, DNAviruses (single and double stranded DNA viruses), RNA viruses (singleand double stranded RNA viruses), point mutations (any organism), singlenucleotide polymorphism (any organism), mRNA's, rTNAs or a micro RNA.15. The biosensor according to claim 14 wherein said bioagent is asingle or double stranded DNA virus.
 16. The biosensor according toclaim 14 wherein said bioagent is a single or double stranded DNAmolecule.
 17. A method of identifying the presence of a bioagent in asample with the ligand based biosensor according to claim 1, said methodcomprising establishing a surface acoustic wave on the surface of saidpiezoelectric material comprising a ligand which binds to said bioagent,exposing said surface to a sample suspected of containing said bioagentand then determining whether the sample contains a suspect bioactiveagent if the wave at the surface of said piezoelectric materialevidences a change consisting with the binding of said bioactive agent.18. The method according to claim 17 wherein said sample is anenvironmental or human sample.
 19. The method according to claim 18wherein said human sample is blood, serum, plasma, urine, sputum orfecal matter.
 20. The method according to claim 17 wherein said surfaceacoustic wave has a specific resonance frequency ranging from 80 to 400Mhz.
 21. The method according to claim 17 wherein said surface acousticwave has a specific resonance frequency of about 325 Mhz.
 22. The methodaccording to claim 17 wherein said bioagent or analyte is selected fromthe group consisting of viruses and their extracts, prions and theirextracts, eucaryotic cells, fragments and extracts, prokaryotic cells,fragments and extracts, prokaryotic spores, fragments and extracts,protein and peptide markers, serum proteins, isolated proteins,synthethic chemicals, viral membranes, eukaryotic membranes, eukaryoticcell parts, including mitochondria and other organelles, prokaryoticmembranes, DNA viruses (single and double stranded DNA viruses), RNAviruses (single and double stranded RNA viruses), point mutations (anyorganism), single nucleotide polymorphism (any organism), mRNA's, rTNAsor a micro RNA.
 23. A ligand based biosensor comprising a lithiumtantalate material and at least one biological ligand complexed,directly or indirectly to the surface of said material, said materialfurther comprising at least two delay lines wherein at least one of saiddelay lines is a reference delay line which produces a surface acousticwave at the surface of the biosensor and at least one of said delaylines is a test delay line which measures the frequency of the acousticwave at the surface of the biosensor to detect the binding of a bioagentor analyte to said ligand. 24-46. (canceled)
 47. A biosensor comprising:a housing; at least one first transducer disposed in said housing forgenerating surface acoustic waves in a fluid disposed in said housing incontact with said first transducer; a plurality of second transducersdisposed in said housing for detecting said surface acoustic waves, saidfluid also being disposed in contact with said second transducers; abiological ligand complexed, directly or indirectly at least to asurface disposed in said housing so as to be in contact with said fluid;an electric circuit operatively linked to said first transducer and saidsecond transducers for operating said first transducer to generate saidsurface acoustic waves at a predetermined frequency and for monitoringsignals from said second transducers to detect changes in said surfaceacoustic waves owing to a binding of a bioagent or analyte to saidligand, thereby detecting the presence of said bioagent or analyte. 48.The biosensor defined in claim 47 wherein said first transducer and saidsecond transducers are inter-digital electrodes disposed on said surfacein said housing, at least said second transducers being connected torespective output delay lines.
 49. The biosensor defined in claim 47wherein said surface is a surface of a substrate made at least in partof piezoelectric material.
 50. A method of identifying a bioagent,comprising: providing a housing having a surface to which a ligand isdirectly or indirectly complexed; generating a surface acoustic wave ina fluid disposed in said housing in contact with said surface; exposingthe fluid in said housing to a sample containing a bioagent which bindsto said ligand; automatically monitoring the surface acoustic wave afterthe exposing of said fluid to said sample; and detecting a change insaid surface acoustic wave thereby determining a binding of saidbioagent to said ligand and according the presence of said bioagent insaid sample.
 51. The method defined in claim 50 wherein the generatingof said surface acoustic wave comprises transmitting a signal to a firstinter-digital electrode disposed in said housing in contact with saidfluid.
 52. The method defined in claim 50 wherein the monitoring of thesurface acoustic wave comprises operating a plurality of inter-digitalelectrodes in contact with said fluid.